FIGURE 1.

Epithelial Hydra piRNAs have sequence signatures typical of ping-pong biogenesis. (A) Schematic of the Hydra body plan and cell types adapted from Siebert (2018). Hydra is comprised of three cell lineages. The two epithelial cell lineages, endoderm (end, green) and ectoderm (ect, blue), form two monolayers separated by a basal lamina called the mesoglea. The cells of the interstitial lineage (pink) are embedded among the epithelial cells; this lineage is supported by a multipotent ISC which gives rise to three somatic cell types: neurons, gland cells, and nematocytes. Interstitial cells shown: progenitors (prog), nematoblasts (nb), nematocyte (nem), ganglion neuron (gn), sensory neuron (sn), and zymogen gland cell (zmg). The ISCs are also able to produce GSCs (not shown). In contrast, the epithelial cell lineages are strictly somatic. The epithelial cells of the body column (shown in lighter colors) are mitotically active, unipotent stem cells that express PIWI proteins (Juliano et al. 2014). Due to mitotic divisions, these cells are translocated toward the oral and aboral ends of the animal. When epithelial cells arrive at the extremities (shown in darker colors), the cells exit the cell cycle, lose PIWI expression, and terminally differentiate to build the hypostome and tentacles at the oral end and the peduncle (ped) and basal disc (bd) at the aboral end. (B) Experimental design for Figures 1, 2: piRNAs were extracted from either untreated (wild-type, contains germline-competent interstitial lineage) or colchicine-treated (epithelial, does not contain interstitial cells) Hydra by immunoprecipitation (IP) using Hywi- and Hyli-specific antibodies (Juliano et al. 2014). piRNAs were isolated from immunoprecipitated complexes, sequenced, and mapped to the Hydra transcriptome (Siebert et al. 2019). (C–J) The number of occurrences of complementary overlap between the 5′ ends of Hywi- and Hyli-bound piRNAs; all four possible combinations were tested. “S” indicates piRNAs that map in a sense orientation to a transcript, and “AS” indicates piRNAs that map in an antisense orientation to a transcript. The high frequency of 10 bp overlap between Hywi- and Hyli-bound piRNAs is indicative of ping-pong biogenesis. This signal is present in piRNAs isolated from both wild-type (WT) (C–F) and epithelial (G–J) Hydra, indicating that the ping-pong biogenesis pathway is active in ESCs. The highest frequency of 10 bp overlaps occurs between antisense Hywi piRNAs and sense Hyli piRNAs in both WT (C) and epithelial Hydra (G). This is consistent with a previous study analyzing piRNAs isolated from WT Hydra vulgaris strain 105 (Juliano et al. 2014).